Yu Man Him. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 45-50). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgement --- p.ii / Chapter 1. --- Introduction / The mitochondrion --- p.1 / The mitochondrial genome --- p.3 / Mitochondrial DNA and diseases --- p.7 / Circulating plasma DNA --- p.8 / Project aims --- p.8 / Chapter 2. --- Materials and Methods / Choice of gene quantification system --- p.9 / Real time quantitative PGR --- p.11 / 7700Sequence Detection System --- p.14 / The LightCycler ´ؤ an alternative fast analyzer --- p.15 / Quatntitation of starting copy number using real time PCR --- p.17 / Primer and probe design rules --- p.18 / Hot start PCR technique --- p.19 / Other anti-contamination measures --- p.20 / Test subjects --- p.21 / Sample processing --- p.23 / DNA extraction --- p.24 / Chapter 3. --- System Development / Choice of primer and probe sequences --- p.28 / Optimization of PCR conditions --- p.30 / Imprecision of TaqMan assays --- p.33 / MTRNR2 vs. MTCYB probes --- p.33 / Construction of standard curve --- p.35 / Chapter 4. --- Research Application / The trauma model --- p.37 / Plasma DNA as a marker for trauma severity --- p.37 / Results --- p.38 / Disussion --- p.41 / Chapter 5. --- Conclusion --- p.44 / Chapter 6. --- References --- p.45

DNA polymerases play a fundamental role in the transmission and maintenance of genetic information and have become an important in vitro diagnostic and analytical tool. The Loop-mediated isothermal DNA amplification (LAMP) method has major applications for disease and pathogen detection and utilises the unique strand-displacement activity of a small group of thermostable DNA polymerases. The Large (Klenow-like) Fragment of Geobacillus stearothermophilus DNA polymerase I (B.st LF Pol I) currently serves as the enzyme of choice for the majority of these isothermal reactions, with few alternatives commercially available. An increasing need for point-of-care nucleic acid diagnostics is now shifting detection methods away from traditional laboratory based chemistries, such as the polymerase chain reaction (PCR), in favour of faster, and often simpler, isothermal methods. It was recognised that in order to facilitate these rapid isothermal reactions there was a requirement for alternative thermostable, strand-displacing DNA polymerases and this was the basis of this thesis. This thesis reports the successful identification of polymerases from Family A, chosen for their inherent strand-displacement activity, which is essential for the removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis in vivo. Twelve thermophilic organisms, with growth temperature ranges between 50oC and 80oC, were identified and the genomic DNA extracted. Where DNA sequences were unavailable, a gene-walking technique revealed the polA sequences, enabling the Large Fragment Pol I to be cloned and the recombinant protein over-expressed in Escherichia coli. A three-stage column chromatography purification permitted the characterisation of ten newly identified Pol I enzymes suitable for use in LAMP. Thermodesulfatator indicus (T.in) Pol I proved to be the most interesting enzyme isolated. Demonstrating strong strand-displacement activity and thermostability to 98oC, T.in Pol I is uniquely suitable to a newly termed heat-denaturing LAMP (HD-LAMP) reaction offering many potential advantages over the existing LAMP protocol. The current understanding of strand-displacement activity of Pol I is poorly understood. This thesis recognised the need to identify the exact regions and motifs responsible for this activity of the enzyme, enabling potential enhancements to be made. Enzyme engineering using site-directed mutagenesis and the formation of chimeras confirmed the importance of specific subdomains in strand-separation activity. With this knowledge, a unique Thermus aquaticus (T.aq) Pol I mutant demonstrated sufficient strand-displacement activity to permit its use in LAMP for the first time. The fusion of Cren7, a double-stranded DNA binding protein, to Pol I for use in LAMP is also reported. Although the fusion construct was found to reduce amplification speed, enhancements were observed in the presence of increased salt concentrations and it is suggested here as a means for future enzyme development.

The Spt4-Spt5 heterodimer complex is one of the key transcription elongation factors for RNA polymerase II, and thus helps to regulate gene expression with either positive or negative stimulation. Spt5 is a part of the NusG family of proteins, and is universally conserved across all three domains of life. This complex is also noted to be involved in many other cellular functions, including chromatin folding, DNA repair, and 5’ cap recruitment; both subunits also play roles in cellular activity when not bound together. However, there is still a great deal of insight to gain about this compound’s functions. This report delves into a variety of previous studies on this complex, summarizing known facts. It will describe how the Spt4-Spt5 complex is actually involved in facilitating transcription for nearly every type of RNA polymerase known so far, and that the secondary characteristics define each homologous structure. The variety of laboratory techniques utilized in these studies will also be noted, and the functionality of this versatile complex will be conveyed as known.

O interesse da Medicina Veterinária nas espécies silvestres tem aumentado gradativamente, principalmente no estudo dos contextos ecológicos de saúde. Dentro desse contexto, autores realizaram estudos com o objetivo de conhecer a importância de Salmonella sp. na saúde das aves silvestres e seu potencial de transmissão para humanos e outros animais. Informações sobre a prevalência e distribuição dos sorovares de salmonelas na população de animais silvestres e domésticos são essenciais para relacionar os possíveis reservatórios que possam ser responsáveis pela transmissão dessa zoonose. Este trabalho teve como objetivo a detecção de Salmonella sp. em psitacídeos clinicamente sadios por Reação em Cadeia da Polimerase (PCR). Foram coletados suabes cloacais de 280 psitacídeos mantidos em cativeiro no Estado do Rio Grande do Sul, pertencentes a treze espécies, provenientes de um zoológico, um criadouro conservacionista e um criadouro comercial. O DNA das amostras foi extraído pelo método de fenol-clorofórmio e examinados pela PCR com a utilização de um par de iniciadores que amplifica um fragmento de 284 pb do gene invA pertencente ao gênero Salmonella, resultando em 37 amostras positivas. Não houve diferença na prevalência de salmonela entre os três plantéis nem entre as 13 espécies analizadas. Não foi possível a detecção desse patógeno pela PCR com iniciadores para a identificação de S. Typhimurium, S. Enteritidis, S. Pullorum e S. Gallinarum, nem através da Técnica Microbiológica Convencional nas amostras detectadas pela PCR genérica, provavelmente devido a maior sensibilidade e especificidade da PCR genérica. De acordo com a revisão bibliográfica realizada, este foi o primeiro trabalho de detecção direta de Salmonella em psitacídeos utilizando a PCR. Os resultados indicaram que aproximadamente 13,2% dos psitacídeos mantidos em cativeiro eram portadores assintomáticos ou eram transientemente infectados pelo gênero Salmonella.

No description available.

One major pathway to overcome DNA damage induced replication blocks is translesion DNA synthesis, which is the replicative bypass of DNA damage by non-classical polymerases. For the cell to utilize translesion synthesis the non-classical DNA polymerase is recruited to sites of DNA damage, and a polymerase switch occurs between the stalled classical polymerase and the incoming non-classical polymerase. This process requires the replication accessory factor proliferating cell nuclear antigen (PCNA) and its monoubiquitination at Lys-164. To better understand the role of PCNA during translesion synthesis, I biochemically and structural characterized two PCNA mutant proteins, G178S and E113G PCNA, which are defective in translesion synthesis. The X-ray crystal structure of both mutant proteins showed a shift in an extended loop, called loop J, compared to the wild type PCNA structure. Steady state kinetic studies determined that in contrast to wild type PCNA which stimulates the non-classical polymerases, the two PCNA mutant proteins fail to stimulate the activity of the non-classical polymerase pol η. These results indicate that loop J in PCNA plays an essential role in facilitating translesion synthesis. During the structural studies of the E113G PCNA mutant protein I observed a unique PCNA structure that failed to form the characteristic PCNA ring shape structure, through traditional intersubunit interactions of domain A and domain B on neighboring subunits. Instead this non-trimeric PCNA structure formed A-A and B-B intersubunit interactions. The B-B interface is structurally similar to the A-B interface observed for the trimeric ring shaped form. In contrast the A-A interface is stabilized by hydrophobic interactions. The location of the E113G substitution is directly within this hydrophobic surface and would not be favorable in the wild type protein. This suggests that the side chain of Glu-113 promotes trimer formation by destabilizing these possible alternate subunit interactions. To biochemically and structurally characterize the impact of monoubiquitinating PCNA (Ub-PCNA), I developed an Ub-PCNA analog by splitting the protein into two self-assembling polypeptides. This analog supports cell growth and translesion synthesis in vivo, and steady state kinetic studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol η in vitro. The X-ray crystal structure of Ub-PCNA showed that the ubiquitin moieties are located on the back face of PCNA. Surprisingly, the attachment of ubiquitin does not change PCNA's conformation. This implies that PCNA ubiquitination does not cause an allosteric change to PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases.

Das Polyomavirus WU (WUPyV) wurde erstmalig im Jahr 2007 in respiratorischem Material bei Patienten mit respiratorischem Infekt beschrieben. Charakterisierung, Epidemiologie und Beurteilung des Krankheitswerts des neuen Virus sind seither Gegenstand vieler Studien weltweit. Retrospektiv wurde Probenmaterial aus dem Respirationstrakt auf WUPyV mittels PCR untersucht. Das Material war zur virologischen Routinediagnostik eingegangen und stammte von in der Universitätskinderklinik Würzburg stationär behandelten Kindern, deren klinische Diagnosen anonymisiert zur Verfügung standen. Es wurden 1277 Nasenrachensekrete (NRS) berücksichtigt aus dem Zeitraum zwischen Januar 2002 und September 2005 sowie zwischen Januar und Juli 2007. Von 1277 NRS waren 62 (4,9 %) positiv für WUPyV. Das Virus wurde in jedem Monat eines Jahres nachgewiesen, wobei Wintermonate insgesamt stärker vertreten waren. Das mediane Alter der betroffenen Patienten betrug 3,0 Jahre (4 Monate – 6,3 Jahre). Klinische Diagnosen bei WUPyV-Infektionen umfassten ein breites Spektrum an oberen und unteren Luftwegserkrankungen. Bei 33 NRS (53,2 %) waren neben WUPyV zuvor ein oder zwei weitere respiratorische Viren durch PCR oder Immunfluoreszenz-Antigentest nachgewiesen worden (Adenovirus: 10; Influenza A: 10; humanes Bocavirus: 9; RSV: 5; Parainfluenzavirus 1/2/3: 3). Die Sequenzanalyse eines 647 bp langen Abschnitts der nicht kodierenden Region bei 50 WUPyV-positiven NRS zeigte eine Übereinstimmung der Sequenzen von 98,5 %. Die Ergebnisse der vorliegenden Studie unterstützen bisherige Ergebnisse zur Epidemiologie und Verbreitung des WUPyV. Demnach konnte WUPyV-DNA bei akuter respiratorischer Infektion im menschlichen Respirationstrakt, bevorzugt bei Kleinkindern, detektiert werden. WUPyV wies eine hohe Koinfektionsrate mit anderen respiratorischen Viren auf. Es zeigte sich in der phylogenetischen Analyse zweier Genomabschnitte eine geringe Variabilität des Genoms. Bei bisheriger Datenlage bleibt unklar, ob der Nachweis von WUPyV-DNA in NRS mit einer akuten respiratorischen Erkrankung assoziiert werden kann. / WU Polyomavirus (WUPyV) was identified in 2007 in respiratory tract samples from individuals with acute respiratory tract infection. Since then there was research worldwide to characterize the new virus in terms of epidemiology, prevalence as well as virus pathogenicity, associated diseases and clinical aspects. We tested 1277 nasal pharyngeal aspirates (NPA) for WUPyV-DNA with a qualitative PCR. NPA were collected from patients, which were treated for acute respiratory tract infection at the University Children’s Hospital in Wuerzburg, Germany. The samples were sent to the Institute of Virology and Immunobiology, Wuerzburg, for routine screening of respiratory viruses between January 2002 and July 2007. Clinical diagnoses were available for retrospective analyse. We found 62 out of 1277 (4,9%) NPA positive for WUPyV-DNA. The virus circulated during the whole year. The median age of infected patients was 3 years (range 4 months to 6,3 years). Clinical diagnoses of the patients included symptoms of upper and lower respiratory tract infections. Co-infections with other respiratory viruses occurred in 33 NPA (53,2%), simultaneously detected with WUPyV were Adenovirus, Influenza A-Virus, human Bocavirus, RS-Virus and Parainfluenzavirus 1/2/3. Additionally we analysed in a set of 50 WUPyV-positive samples a 647 bp sequence of the non-coding control region and revealed a sequence identity of 98,5%. The results of this study were able to reproduce data of previous reports concerning the newly discovered WUPyV. The virus was detected in NPA of patients with acute respiratory tract infection worldwide, particularly in children at the age of 4 and younger. It goes along with a high co-infection rate with other respiratory viruses. In phylogenetic analyses the WUPyV genome seems to have a high sequence identity. Further studies need to be done to determine the pathogenicity of WUPyV and connect the presence of the virus in human samples to a causal relationship with a specific clinical condition.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1997. / Includes bibliographical references. Also available on the Internet.

Rayleigh-Bénard convection has been extensively studied in literature owing to its ubiquitous nature. However, most of the studies have been confined to geometries where the aspect ratio of the cylinder was less than 1. Here we study the motion of fluid in geometries with aspect ratio greater than 1, with particular application to use of such motion to actuate biochemical reactions, such as the polymerase chain reaction. We show that it is possible to accelerate the rate of reaction by using a geometry that promotes chaotic motion versus a geometry that promotes quasi- periodic motion. We also simulate chemical kinetics using the fluid motion as a starting point and we prove that chaotic motion indeed enhances the rate of the reaction. We also provide qualitative and quantitative measures for chaotic motion in a fluid flow, which helps to distinguish between different types of fluid motion. We highlight the transitions between different types of flow that are possible with Rayleigh-Bénard convection. Finally, we compare our simulations against experimental data obtained from particle image velocimetry, laser induced fluorescence and optical microscopic visualization.

Zugl.: Seattle (Wash.), Univ. of Washington, Diss., 2007 / Hergestellt on demand